Medical treatments are often highly generalized across patients with a particular disease. Treatment and drug efficacy become hampered as a result due to inherent heterogeneity between patients and between diseases. Therefore, to optimize therapeutic effects and minimize hazardous side effects, a personalized approach to medicine, involving individually tailored treatment plans (i.e. type and dosage of therapeutic), is desired. This requires preliminary tests, known as companion diagnostics, to assess the efficacy of a drug prior to administration. For cancer treatment, there is a growing interest in targeting differential enzymatic activity with prodrugs (precursor forms activated by an intracellular enzyme). Understanding enzymatic activity of the tumor prior to prodrug administration will allow physicians to design a better therapeutic plan. Currently, however, there exists no such method. Therefore, as a tool for personalizing cancer treatment, we have developed a novel, robust technology to accompany all prodrug treatments. This technology, termed P-MED (Personalized Monitoring of Enzyme Dynamics), provides quantitative enzymatic activity measurements of single cells in a high-throughput manner by monitoring the turnover of fluorogenic substrate in a microfluidic device. Cells are manipulated using inertial effects that occur on the microscale to allow synchronized measurements of fluorescence accumulation that report on enzymatic activity. We have used this system to measure intracellular esterase activity (currently targeted for colorectal cancer therapy) in proof-of-concept model cells (Jurkat and HeLa cells). Additionally, P-MED can monitor the response to enzymatic inhibitors (nordihydroguaiaretic acid). P-MED can be used to predetermine the success of a variety of drugs on a perpatient basis. This method has far reaching implications in cancer treatment and pharmaceutical drug testing.